Separation of macromolecules by gel electrophoresis is a well established technique. The technique has demonstrated utility for the separation of various types of biomacromolecules such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides, proteins, peptides, etc. and permits separation of mixtures of these materials into isolated, discrete bands for analytical or preparative purposes. A basic requirement of the electrophoretic gel is that it must have a porous network through which the charged analyte biomolecules can migrate; because of differences in size of the macromolecule of varying molecular weight, species of differing sizes migrate through the gel with differing velocities. Large molecules experience more difficulty traversing the porous network presented by the gel and thus move with lower velocities relative to smaller molecules which can more easily penetrate the pores of the matrix and thus move with greater velocities relative to larger molecules. Numerous types of materials are useful for preparing the gel matrix. One requirement of materials used to form gels is that it must form a stable porous matrix, incorporating a large amount of water in the pores of the matrix.
Gel electrophoresis can be performed in numerous formats. These include open horizontal (submarine) slab and closed vertical slab gel formats as well as a cylindrical tube gel format. In any of these formats, the steps for performing an electrophoretic analysis consist of preparation of the gel in a casting container characteristic of the particular format, followed by application of the sample to be analyzed to the gel. Subsequent application of a constant electrical field (either constant voltage, current or power) across the gel causes differential migration of the components of the sample through the porous network formed by the gel to separate into discrete bands of essentially pure components. The separated component bands are then visualized by removing the gel (either tube gel or slab gel) from its original casting container and immersion of the gel into some visualization reagent such as Coomassie Blue stain, silver stain, ethidium bromide, etc.; these visualization agents are chosen to have a differential affinity for the sample components of interest relative to the polymeric material used to form the gel and thus the separated bands preferentially interact with the staining agent.
In some cases, it is necessary to destain the gel by immersing it in another reagent solution which extracts the background staining agent from the bulk of the gel while retaining the staining agent adsorbed or deposited onto the separated sample component bands. As a final step, a permanent record is made of the separation pattern by either removing the water from the gel to yield a dry, transparent record of the gel which can be photographed, autoradiographed, or used as is for a permanent record.
In all of these previous cases, the gel is cast, used for analysis, removed from casting vessel, chemically treated for visualization of the separation, and then dried. Because of the manipulations and treatments with various reagent solutions, it is not possible to reuse the gel for multiple consecutive analyses. Stability and reuse of classical electrophoretic gels is thus rarely an issue and gels used in these formats are treated as single-use, disposable items.
In the classical formats of gel electrophoresis, the electrophoretic separation is performed under a controlled and constant electrical field. Typically, an electrical field is applied across the gel and controlled by maintaining either the applied potential, the current or the power (arithmetic product of voltage and current) constant for the duration of the separation. In recent years, periodic inversion of the electrical field across agarose slab gels has been implemented [G. F. Carle, M. Frank, M. V. Olson, Science, (1986) 232 65-68] as a way of permitting enhanced resolution in the separation of very large (chromosomal size) DNA molecules on such gels. This field inversion was performed to facilitate reorientation of these large molecules in the gel during periods of field inversion and thus prevent separation of the molecules through the gel which would prevent separation of such large molecules. Carle et al. discloses a simple-use gel format.
In recent years, an extension of the tube gel format of electrophoresis has evolved into the use of small diameter (capillary) tube gels. Originally described by U. Grossbach [Biochim. Biophys. Acta, 107 (1965) 180-182] and improved upon in recent years by S. Hjerten [J. Chromatogr. (1983) 270 1-6] and B. Karger et al. [U.S. Pat. Nos. 4,865,706, 4,865,707, 4,997,537], these gels offer numerous advantages over the older, thick-gel formats mentioned above. A significant improvement in the development of capillary gels, which evolved from free zone electrophoretic separations [J. Jorgenson, K. D. Lukacs, Anal. Chem. (1981) 53 1298-1302], is the use of on-line detection technologies for visualization of the separated analyte bands. Thus, with the development of capillary gel electrophoresis, reusability of the gel for multiple separations became desirable for several reasons. Firstly, the gel is never removed from its casting vessel or treated with visualization agents to detect the separated bands. A section of the capillary gel is used as an absorbance or fluorescence cell in the optical path of the appropriate detector which detects all components which migrate by the detector. In practice, reuse of the gel for multiple, consecutive analyses is possible. This permits the gel elution pattern to be calibrated against an appropriate standard mixture. Thus, the gel could be reused for multiple determinations of molecular weights of proteins or the base (or base pair) size of oligonucleotides, RNA and single or double-stranded DNA.
An object of this invention is to provide an improved protocol for extending the utility of electrophoretic gels in general, and capillary electrophoretic gels in particular, thus permitting a greater number of analyses to be achieved on a single gel.
Another object is to improve the analytical precision of capillary electrophoretic gels by reducing the run-to-run variation in migration times for analyte bands to reach the detector.